Grass endophytes

ABSTRACT

The present invention relates to a combination of a  Neotyphodium  species endophyte or endophyte culture in a symbiotic association with a host grass which association does not cause symptoms of toxicosis in animals through exclusion of ergovaline but, due to the inclusion of alkaloids including agroclavine, setoclavine or isosetoclavine, retains the ability to resist abiotic stresses and protect the grass from pests.

TECHNICAL FIELD

This invention relates to fungal endophytes and combinations ofendophytes with grass plants. More particularly the invention relates toendophytes which form combinations with tall fescue (Festucaarundinacea) and some other related grasses. Even more particularly theinvention relates to combinations having reduced toxicity to grazinglivestock as compared to cultivars of endophyte/tall fescue combinationsin common use.

BACKGROUND ART

Fungal endophytes of the genus Neotyphodium (formerly Acremonium) infecta number of temperate climate Pooideae grasses. The Neotyphodiumendophytes can produce alkaloids which are considered to confer degreesof pest and possibly disease protection upon the plants in which theynaturally occur (Rowan and Latch, 1994; Blank and Gwinn, 1992). TheNeotyphodium endophytes are vertically transmitted through the seed ofthe grasses and no natural horizontal transmission has been established(Leuchtmann,1997).

Many of the predominating natural endophyte infections of improved grasscultivars used for pastoral agriculture production also causesignificant animal disorders, for example tall fescue toxicoses(Stuedemann and Hoveland, 1988) and ryegrass staggers (Fletcher et al.,1999). These may be complex toxic reactions by animals to alkaloidsproduced under a range of plant growth conditions. Significant economicloss within pastoral agriculture systems can occur due to such animaltoxicoses. On the other hand presence of at least some endophytes may beessential for the competitive persistence of the chosen grass in apasture (Elberson and West, 1996, Fletcher and Easton, 2000).

Grass lines can be artificially infected with selected endophytes.Axenic cultures of endophytes can be used to infect grass seedlings,grown initially under sterile conditions (Latch and Christensen, 1985),which are then selected for desirable qualifies, and multiplied forcommercial use. Three significant examples of this technology have beendeveloped by the Grasslands division of AgResearch Ltd: GREENSTONE™tetraploid hybrid ryegrass with ENDOSAFE™ endophyte (Tapper and Latch,1999, NZ Patent 233083); various perennial and hybrid ryegrasses withAR1 endophyte (Fletcher and Easton, 2000); and tall fescue cultivarswith MaxQ™ endophyte (Bouton, 2000; Bouton et al., 2002, U.S. Pat. No.6,111,170).

Fescue Toxicosis

Fescue toxicosis has been associated with the natural infection of tallfescue by common strains of Neotyphodium coenophialum. These strainstypically produce the ergopeptine alkaloid, ergovaline, which is of aclass of ergopeptines known to be toxic to mammals. Ergovaline isconsidered to be the primary cause of fescue toxicity. Other compounds,notably other ergoline and ergolene compounds, for example lysergicacid, possibly add to the syndrome (Oliver, 1997; Gadberry et al., 1997;Hill et al., 2001).

The ergovaline levels tend to be higher in leaf sheath and heads of tallfescue than in leaf blade and undergo seasonal variation (Rottinghaus etal., 1991). There is very little ergovaline in roots. Typically aconcentration of ergovaline in herbage or herbage products such as hay,straw, seed or silage of greater than an average of 0.4 ppm of drymatter has been considered a risk of causing fescue toxicosis(Tor-Agbidye et al., 2001) especially when combined with climaticconditions exacerbating fescue toxicosis symptoms.

Other Compounds Recognised as Plant Defence Mechanisms—Peramine, Lolines

Peramine is produced in endophyte-infected grass (Rowan et al., 1986)and probably mobilised within the plant. It is a potent feedingdeterrent for a range of insects, e.g. Argentine stem weevil(Listronotus bonariensis), (Rowan and Latch 1994) and a significantfactor for protecting endophyte-infected grasses from insect pestpredation.

Lolines (N-formylloline, N-acetylloline, N-acetyinorloline and otherclosely related compounds) are produced by some Neotyphodium endophytesincluding N. coenophialum typical of tall fescue. These compounds inappropriate endophyte-infected grasses have properties of deterring orresisting a number of insects, notably sucking insects, for example,Rhopalosiphum padi (Seigel et al., 1990).

Protective Effects in Tall Fescue Pastures—Persistence under StressConditions

Endophyte infection has been associated with enhanced persistence oftall fescue plants under water deficit or drought conditions. Whetherthis effect is due to better resistance of biotic stress factorsexpressed in water deficit situations; general better health ofendophyte-infected tall fescue plants (particularly of root systems); ordue to specific differential physiological responses of theendophyte-infected plants to water stress; is not clearly evident.However, the overall effect is enhanced resistance to water deficit.

Tremorgens

Some Neotyphodium endophytes, notably those of evolutionary derivationfrom strains of Epichloë festucae such as N. lollii, produce potenttremorgens which are toxic to grazing animals. To ensure such tremorgensare not produced by an endophyte artificially introduced into foragetall fescue or ryegrass, the presence of the known potent tremorgenstypical of endophytes, that is the lolitrems, is tested for. Alsotremorgenic activity in grazing test animals is looked for.

It is an object of the present invention to provide an endophyte whichcan produce ergovaline and some ergoline and ergolene compounds at thebase of the tall fescue leaf sheaths and in the crown of the plant butonly in a manner such that the usual concentration in herbage asgenerally consumed by grazing animals in common farming practice is lessthan a practical threshold toxicity level.

For the purposes of this specification “crown” is defined as that areaof a grass plant which is generally less than 2 cm above soil level andexcludes the roots of the grass, but includes the base of tillers andlateral meristem growing points for new vegetative tillers.

It is a further object of the invention to provide an endophyte whichcan produce lolines in amounts which are considered to be partially orsubstantially effective in deterring some insect pests from feeding onplants.

It is a further object of the invention to provide an endophyte whichdoes not produce detectable levels of lolitrems and are not observablytremorgenic.

It is a still further object of the present invention to address theforegoing problems or at least to provide the public with a usefulchoice.

All references, including any patents or patent applications cited inthis specification are hereby incorporated by reference. No admission ismade that any reference constitutes prior art. The discussion of thereferences states what their authors assert, and the applicants reservethe right to challenge the accuracy and pertinency of the citeddocuments. It will be clearly understood that, although a number ofprior art publications are referred to herein, this reference does notconstitute an admission that any of these documents form part of thecommon general knowledge in the art, in New Zealand or in any othercountry.

It is acknowledged that the term ‘comprise’ may, under varyingjurisdictions, be attributed with either an exclusive or an inclusivemeaning. For the purpose of this specification, and unless otherwisenoted, the term ‘comprise’ shall have an inclusive meaning—i.e. that itwill be taken to mean an inclusion of not only the listed components itdirectly references, but also other non-specified components orelements. This rationale will also be used when the term ‘comprised’ or‘comprising’ is used in relation to one or more steps in a method orprocess.

Further aspects and advantages of the present invention will becomeapparent from the ensuing description which is given by way of exampleonly.

DISCLOSURE OF INVENTION

According to one aspect of the present invention there is provided anendophyte of Neotyphodium coenophialum species, selected from the groupconsisting of: AR512; AR513; AR514; AR517; AR521; AR522; AR524; AR525;AR535; AR539; and combinations thereof; AR512; AR513; AR514; AR517;AR521; AR522; AR524; AR525; AR535, AR539 being cultures deposited on 2Oct. 2002 at the Australian Government Analytical Laboratories (AGAL)with accession numbers: NM02/31935; NM02/31936; NM02/31937; NM02/31938;NM02/31939; NM02/31940; NM02/31941; NM02/31942; NM02/31943; NM02/31944;

-   -   characterised in that, in combination with a host grass, said        endophyte does not cause symptoms of toxicosis in animals;    -   and further characterised in that the endophyte retains        sufficient levels of at least two alkaloids selected from the        group consisting of: agroclavine; setoclavine; isosetoclavine;        and combinations thereof, that protect the host grass from pests        or abiotic stresses or both;    -   and further characterised in that the host grass is artificially        inoculated with the endophyte.    -   According to a further aspect of the present invention there is        provided an endophyte culture of Neotyphodium coenophialum        species, selected from the group consisting of: AR512; AR513;        AR514; AR517; AR521; AR522; AR524; AR525; AR535; AR539; and        combinations thereof; AR512; AR513; AR514; AR517; AR521; AR522;        AR524; AR525; AR535, AR539 being cultures deposited on 2 Oct.        2002 at the Australian Government Analytical Laboratories (AGAL)        with accession numbers: NM02/31935; NM02/31936; NM02/31937;        NM02/31938; NM02/31939; NM02/31940; NM02/31941; NM02/31942;        NM02/31943; NM02/31944;characterised in that, in combination        with a host grass, said endophyte culture does not cause        symptoms of toxicosis in animals;    -   and further characterised in that the endophyte culture retains        sufficient levels of at least two alkaloids selected from the        group consisting of: agroclavine; setoclavine; isosetoclavine;        and combinations thereof, that protect the host grass from pests        or abiotic stresses or both;    -   and further characterised in that the host grass is artificially        inoculated with the endophyte culture.

Preferably, the toxicosis which is avoided is fescue toxicosis. Mostpreferably the toxicosis is caused by an ergovaline toxin.

Preferably, the level of ergovaline in the present invention is lessthan 0.4 ppm in dry matter in herbage consumed by grazing animals. Morepreferably, the level of ergovaline is less than 0.4 ppm in dry matterin herbage, other than the crown of the host grass plant, consumed bygrazing animals.

Preferably, the abiotic stress is a water deficit.

Preferably, the endophyte culture, if used, is an axenic culture.

Preferably, the endophyte or endophyte culture produces less than 0.2ppm ergovaline in dry matter of whole herbage when infected into hostgrass.

According to a further aspect of the present invention there is provideda combination of the endophyte as described above, and a host grass.

According to another aspect of the present invention there is provided acombination of the endophyte culture as described above, and a hostgrass.

Preferably, the combination, substantially as described above, isachieved by modification of host grass infected with the endophyte orendophyte culture by methods selected from the group consisting of:breeding; crossing; hybridisation; genetic modification; andcombinations thereof.

Preferably, the host grass used in the combination described above isselected from the group consisting of: tall fescue grass cultivar,ryegrass culivar, meadow fescue cultivar, and combinations thereof.

According to a further aspect of the present invention the host grass isa Pooideae grass.

According to a further aspect of the present invention there is provideda combination of endophyte or endophyte culture, as described above, anda host grass wherein the combination produces isosetoclavine andsetoclavine at a rate of greater than 0.5 ppm each of dry matter in thehost grass plant crowns. Preferably also, the combination produces lessthan 0.2 ppm of dry matter of ergovaline in whole heritage.

According to a further aspect of the present invention there is provideda combination of endophyte as described above and a host grass, whereinthe combination has features selected from the group consisting of:enhancement of pest protection, resistance to insects, pasturepersistence, and combinations thereof.

According to a further aspect of the present invention there is provideda combination as described above and a host grass, wherein thecombination has the features of enhancement of grazing animal growth andincreased animal productivity relative to grass infected with knownendophytes capable of inducing fescue toxicosis.

According to a further aspect of the present invention there is provideda combination as described above and a host grass wherein the pest towhich increased resistance is conferred on the host grass is selectedfrom the group consisting of: lesion nematode, root aphid, corn fleabeetle, and combinations thereof.

According to a further aspect of the present invention there is providedseeds of a host grass infected with the endophyte substantially asdescribed above.

According to yet a further aspect of the present invention there isprovided seeds of a host grass infected with endophyte culture asdescribed above.

The invention is the combination of examples of a class of Neotyphodiumcoenophlalum endophyte and improved grass cultivars by artificialinoculation to produce host grasses which do not cause symptoms oftoxicosis by way of the ergovaline toxin, but which retain sufficientlevels of other alkaloids (for example: agroclavine, setoclavine and/orisosetoclavine) to individually or in combination continue to protectthe host grass from pests or abiotic stresses (such as water deficit) orboth.

The inventon has been achieved by understanding the biology ofendophytes of temperate climate grasses, isolating selected endophytesof interest, inoculating the endophytes into surface-sterilisedseedlings of grasses, exemplified by improved tall fescue or perennialryegrass cultivar lines, re-evaluating alkaloid production, multiplyingseed, evaluating for agronomic factors, testing for animal production,evaluating for any evidence of animal disorders such as fescuetoxicosis, staggers, hyperthermia, or prolactin hormone depression andtesting for invertebrate pest protection.

The invention consists of the foregoing and also envisages constructionsof which the following are examples.

BEST MODES FOR CARRYING OUT THE INVENTION

Culture Conditions and Description

All endophytes of this invention are strains from collections of seed oftall fescue originally sourced from the Claviplus class. Seed fromvarious tall fescue collections were examined for the presence ofendophyte by seed squash technique. A selection of plants for each seedsample, where an endophyte was shown to be present, were grown for a fewweeks in glasshouse conditions and re-tested for endophyte presence intheir leaf sheaths.

The endophytes from plants with chemotypes of interest were isolated andgrown in culture according to the method of Latch and Christensen(1985). The endophytes of this invention are held in a culturecollection or in cloned plants at the Grasslands site of AgResearch Ltdin Palmerston North, New Zealand. The cultures are also deposited at theAustralian Government Analytical Laboratories in Sydney, Australia.

The endophyte cultures were deposited with the Australian GovernmentAnalytical Laboratories (AGAL), New South Wales Regional Laboratory, 1,Suakin Street Pymble NSW 2073, Australia, on Oct. 2, 2002, under theterms of the Budapest Treaty on the International Recognition of theDeposit of Microorganisms for the Purposes of Patent Procedure. Theywere assigned the following numbers: NM02/31935 (AR512); NM02/31936(AR513); NM02/31937 (AR514); NM02/31938 (AR517); NM02/31939 (AR521);NM02/31940 (AR522); NM02/31941 (AR524); NM02/31942 (AR525); NM02/31943(AR535); and NM02/31944 (AR539). All strains of endophyte of thisinvention are accommodates within a single sub-grouping of the speciesNeotyphodium coenophialum. The isolates, when grown on potato dextroseagar at 22° C., are slow-growing (radial growth approximately 0.1-0.3 mmper day) with colonies typically white and cottony. Conidia have beenobserved at variable rates of production near the margins.

Inoculations

Axenic cultures of endophytes AR512, AR513, AR514, AR517, AR521, AR522,AR524, AR525, AR535, and AR539 as examples of this invention, weresuccessfully inoculated (Latch and Christensen, 1985) into seedlingsgrown from surfaced sterilised seed of the tall fescue cultivar Kentucky31. Endophytes AR514, AR524, and AR525 were also infected into othercultivars such as, for example Grasslands Flecha, Jesup Improved,Georgia 5 and various experimental lines, generally with a satisfactorysuccess rate well in excess of 5% of attempts. No complete failures toinfect tall fescue were observed with the endophytes of this invention.

A typical meadow fescue cultivar (Ensign) was successfully inoculatedwith endophyte AR512. Similarly a typical perennial ryegrass test line(GA66) was successfully inoculated with endophytes AR514 and AR525 forfurther examination with the chemotype characteristics of thecombinations similar as for when infecting tall fescue, but generallywith lower levels of alkaloid accumulation, and with rates of infectionof the order of 1% to 5% of attempts.

Seed has been successfully produced from infected plants containingendophytes of this invention under routine seed production conditionswith relatively high and useful rates of endophyte infection.

Chemotype Identification

Basal parts of endophyte-infected tillers were freeze dried, sometimesmilled, and extracted and analysed qualitatively for the presence ofergovaline by high performance liquid chromatography (HPLC) as set outbelow. Those indicating ergovaline less than approximately 0.2 ppm ofdry matter were further analysed for the production of peramine at ratesgreater than about 2 ppm of dry matter. The endophytes from suchselections were isolated, classified by culture attributes, andgenerally re-inoculated into seedlings of endophyte-free tall fescue,cultivar Kentucky 31, as a typical improved pasture host for comparativepurposes. Samples from such plants at various stages of growth wereanalysed in more detail for alkaloid production, including for theproduction of tremorgenic lolitrems typical of N. lolli infection andlolines typical of N. coenophialum.

The expression of alkaloid production of endophyte-infected tall fescue,using endophytes selected from tall fescue sources, was observed to fallbroadly into three groups. The most numerous group produce bothergovaline and peramine at levels often well in excess of 1 ppm in thebasal tiller material. Such endophytes are likely to be associated withfescue toxicosis of grazing animals if present at high rates ofinfection in forage.

A second chemotype group produced peramine and lolines, but nodetectable trace of ergovaline. Endophytes typical of this group havebeen developed for commercial application (Bouton, 2000; Bouton et al.,2002, U.S. Pat. No. 6,111,170).

A third group, of this invention, which have low levels and acharacteristic distribution within plants of ergovaline, are discussedbelow. They are further characterised by the presence of peramine inherbage generally well in excess of 1 ppm, and the presence of lolinesin herbage in amounts within ranges typical of N. coenophialuminfection. In the course of chromatographic analysis for ergovaline thepresence of other compounds with UV and fluorescence spectral propertiestypical of ergolene derivatives (i.e. fluorescent “ergot alkaloids”)were observed, notably in the basal portions of tillers, crowns, andseed of tall fescue plants infected with this group of endophytes. Thesecompounds are also discussed below.

Lolines (N-formylloline, N-acetylloline, and N-acetylnorloline, in orderof usual observed abundance) were detected by capillary gaschromatography in extracts of tall fescue and meadow fescue plantsinfected with the endophytes of this invention in amounts more or lesscomparable to that observed in comparable tall fescue plants infectedwith common N. coenophialum. The methods used were minor modificationsof the method of Yates et al., (1990).

Lolitrems were not detected by chromatographic analysis in any tallfescue infected with endophytes of this invention. The possible effectsof lolitrems were not further directly considered, other than byobservation of lambs grazing pastures containing endophyte AR514 forsigns of tremors.

Ergot Alkaloid Levels and Identification of New Alkaloids

Ergovaline concentration was measured by HPLC with fluorescencedetection. Typically, a sample of approximately 50 mg of milled (1 mmmesh), freeze-dried endophyte-infected tissue from the lower 3 to 5 cmof tillers from mature vegetative plants (basal tiller materialpredominantly consisting of leaf sheaths) was extracted with 1 ml of amixture of equal parts of propan-2-ol and water containing also 1%lactic acid. The extraction continued for one hour at ambienttemperature with gentle mixing. An internal standard of added ergotaminetartrate (c. 1 μg per sample) was used for quantitative comparisons.

Following brief centrifugation, a sample of the clarified extractsolution was taken for HPLC using a reverse phase column (typicallyProdigy 150×4.6 mm, 5 μm silica ODS (3), Phenomenex, Calif., USA), withelution at 1 ml per minute and a solvent gradient of acetonitrile and100 mM aqueous ammonium acetate by volume starting at 27.5% acetonitrileand progressing in linear stages to 35% at 20 min, 50% at 35 min, 60% at40 min and 75% at 50 min.

Naturally fluorescent ergolene compounds including ergovaline (and Rsisomer ergovalinine), the internal standard ergotamine (and its isomerergotaminine partially formed during extraction) and the compoundsobserved in the earlier section of the chromatograms were detected byusing UV excitation at 310 nm and emission at 410 nm. Ergovaline, itsisomer ergovalinine, added ergotamine internal standard and its isomerergotaminine elute at approximately 22, 36, 30 and 42 minutesrespectively. The minimum detection level for routine analysis ofergovaline (combined amounts with isomer ergovalinine) is approximately0.05 ppm of dry matter.

During the course of examining examples of tall fescue infected withendophytes of this invention for ergovaline, other ergolene derivativeswere observed to be present in a pattern not previous recognised.Compounds eluting at approximately 6.5 minutes (compound A) and 8.0minutes (compound B) are characteristically both present inendophyte-infected basal tiller and crown material of this invention.These compounds have been identified as isosetoclavine and setoclavinerespectively, as follows.

Fractions enriched in compounds A and B were prepared from a methanol-1%aqueous acetic acid (4:1) extract of bulked freeze dried and milledlower sheath of tillers containing endophytes AR514 and AR524(pre-extracted with hexane). The extract was fractionated by sequentialreverse-phase flash chromatography on octadecyl-functionalised silicagel (Aldrich) with acidic (methanol-1% aqueous acetic acid) and neutral(methanol-water) step gradients, and normal-phase flash chromatographyon silica gel (silica gel 60, 40-63 μ, Merck) with an ethylacetate—methanol step gradient

The characteristic fluorescence and the UV absorption spectra andelectrospray ionisaton (ESI) mass spectral data for compounds A and Bobtained by HPLC analysis of enriched fractions using variations of HPLCseparation conditions and detectors (Shimadzu LC-MS instrument QP-5050with SPD-10AVP UV diode array and RF-10A fluorescence spectraldetectors), together with comparative data with standards establishthese compounds are setoclavines (isosetoclavine and setoclavinerespectively). Both compounds show strong fluorescence peaks in the HPLC(λ_(Ex) 310 nm λ_(Em) 410 nm). The UV spectra are characterised bymaximum absorbances at 307 nm and 312 nm respectively and for bothcompounds the positive ion ESI mass spectrum shows a base peak atm/z=237 (MH⁺-H₂O) and an MH⁺ ion at m/z=255. Compound B co-eluted with asample of reference setoclavine provided by Dr Miroslav Flieger,Institute of Microbiology Academy of Sciences of the Czech Republic.Compounds A and B were identical by HPLC, and spectral properties toisosetoclavine (compound A) and setoclavine (compound B) obtained bychemical oxidation of agroclavine by a standard procedure. The presenceof isosetoclavine and setoclavine has not been previously reported inendophyte infected grasses although they have been reported as oxidationproducts of agroclavine in other plant systems (review by Kren, 1999).

The same extracts were also shown to contain agroclavine by electrosprayLC-MS, with an ESI mass spectrum ion attributed to MH⁺ at m/z=239eluting at the same time and with essentially the same UV spectrum asauthentic agroclavine.

Table 1 summarises alkaloid analysis results of specified plant parts oftall fescue infected with the endophytes and usually grown undertemperate summer season conditions, generally in comparative testcultivar Kentucky 31. For consideration of ergot alkaloid production inthe crown and basal tiller, comparison is also made to tall fescueinfected with endophytes AR501 and AR542 which consistently do notappear to produce any ergovaline or setoclavines.

TABLE 1 Examples and typical ranges or scores of alkaloids observed inspecified plant parts Sample (cultivar & Ergovaline Compounds endophyte)Part of plant (ppm DM) A & B Jesup EI Whole herbage 0.4-1.2 ND Jesup EISheath 2.5 Manawatu RS EI Sheath  7.1-15.7 Kentucky 31 EI Whole Herbage1.8-3.0 ND Kentucky 31 EI Leaf blade 0.3-1.8 ND Kentucky 31 EI Sheath 2.9-16.2 Kentucky 31 AR501 Crown <0.1^(ND) * Kentucky 31 AR514 Wholeherbage <0.1 * Kentucky 31 AR514 Crown 0.1-0.6 ** Kentucky 31 AR514Immature heads <0.1-0.2   * Kentucky 31 AR522 Stem & sheath 0.1-0.2 *Kentucky 31 AR522 Crown 3.4-6.1 *** Kentucky 31 AR524 Crown 0.3-0.6 **Kentucky 31 AR524 Immature heads <0.1 * Kentucky 31 AR525 Whole herbage<0.1 ND Kentucky 31 AR525 Crown 0.7-1.0 *** Kentucky 31 AR525 Immatureheads <0.1 * Kentucky 31 AR525 Stem, sheath & heads <0.1 * Kentucky 31AR535 Crown 0.4-0.7 *** Kentucky 31 AR535 Stem, sheath & heads <0.1 **Kentucky 31 AR542 Crown <0.1 ND Kentucky 31 AR542 Sheath <0.1 NDCompound A = isosetoclavine Compound B = setoclavine ND = not detected,detection limit of 0.1 ppm DM for ergovaline * = possible trace or lowlevel ** and *** = score of relative abundance observed EI = infectedwith common toxic or wild type endophyteGenotype Characterization of Endophyte

All endophytes discussed above are characterised by DNA “fingerprinting”(selected polymorphic microsatellite loci and/or Arbitrary FragmentLength Polymorphisms (AFLP) technique) as belonging to a sub-group ofNeotyphodium coenophialum.

Samples of about 50 mg fresh or 15 mg dry basal tiller were used for theextraction of DNA using FastDNA kit for plants (Bio 101, Vista,California) by procedures recommended with the kit. Alternativelygenomic DNA was extracted from cultured endophyte (Moon et al., 1999).Microsatellite PCR amplification was performed using primer pairslabelled with fluorescent dyes, B10.1 (5′-TET)/B10.2 and B11.1(5′-HEX)/B11.4, as described by Moon et al., (1999). The apparent sizeof microsatellite PCR fluorescent labelled products was measuredrelatively to within an estimated 0.3 nucleotide units by capillaryelectrophoresis using an ABI 3100 Genetic Analyzer with POP6 polymerchemistry in 50 cm capillary arrays and GeneScan-400HD standards(Applied Biosystems Inc., Foster City, Calif.). The apparent sizes ofPCR products by this technique (adjusted by subtracting a unit where anadenine nucleotide appears to have been terminally added) are set out inTable 2.

Table 2 shows that the endophytes of this invention can be distinguishedfrom other groups of Neotyphodium endophytes by the number of allelesobserved and the apparent sizes of such alleles. Thus all strains ofthis invention share a B11 allele of size c. 128 base pairs and a secondB11 allele within the size range c. 192 to 200 bp. They also share withother N. coenophialum strains up to three B10 alleles within the rangec.154 to 185 bp.

The presence of three alleles for the endophytes of this invention forthe B10 locus is consistent with evidence for N. coenophialum as ahybrid endophyte derived from three different Epichloë source species(Tsai et al., 1994).

TABLE 2 Apparent size of B10 and B11 microsatellite PCR products. B10locus B11 locus No of No of Source material alleles Allele sizes (bp)alleles Allele sizes (bp) N. coenophialum, wild type, 3 160.4, 169.6,184.2 2 147.9, 192.2 Australian C1, coB isozymes N. coenophialum, wildtype, 2 160.4, 169.4 2 147.9, 192.2 NZ Tindall's, coB isozymes N.coenophialum, wild type, 3 160.3, 169.3, 184.2 2 147.8, 192.0 NZ RS2 &NZ RS6 N. coenophialum, AR542 2 160.5, 169.6 2 180.4, 192.2 Neotyphodiumsp., FaTG-3 2 169.5, 178.7 1 127.9 strain AR501 Neotyphodium sp., FaTG-32 169.6, 178.7 1 127.8 strain AR506 AR539 3 154.6, 172.5, 178.3 2 127.9,192.2 AR513 3 157.7, 160.5, 178.4 2 128.0, 192.2 AR525 3 157.7, 160.4,178.3 2 128.0, 192.2 AR517 3 163.4, 172.5, 178.2 2 128.0, 192.1 AR521 3163.3, 172.5, 178.2 2 127.9, 192.1 AR512 3 172.6, 178.5, 181.5 2 128.0,192.2 AR514 3 157.8, 160.6, 178.4 2 128.0, 196.2 AR522 3 157.7, 160.5,178.3 2 128.0, 200.1 AR524 3 157.7, 160.5, 178.3 2 127.9, 200.2 AR535 3157.7, 160.5, 178.3 2 128.0, 200.1

Analysis by AFLP (Griffiths et al., 1999) also confirmed that endophyteexamples AR514, AR525 and AR535 of this invention are from a sub-groupwhich can be distinguished from other N. coenophialum endophytes outsidethis sub-group by one or more polymorphic differences, but not manydifferences, from within more than 200 AFLP bands observed to bepolymorphic for the genera Neotyphodium and Epichloë.

Safe Grazing with Endophyte in Tall Fescue Cultivar Kentucky 31

Pastures of tall fescue infected with examples of the endophytes of thisinvention do not induce typical fescue toxicosis in grazing animals.Table 3 shows growth rate of lambs in a trial conducted at Eatonton,Ga., USA for two seasons (21 Apr.-30 Jun. 1997, 2 Apr.-2 Jul. 1998). Thegrowth of lambs on a pasture of Kentucky 31 tall fescue infected withendophyte strain AR514 was essentially the same growth as on equivalentendophyte-free pasture and significantly better than growth on naturallyendophyte-infected pasture using the comparable Jesup cultivar. The wildtype endophyte infection significantly reduced live weight gain (P<0.05)and increased mean body (rectal) temperature.

Gross depression of prolactin in blood is another symptom of fescuetoxicosis. Endophyte strain AR514 did not cause a depression ofprolactin whereas with the wild type endophyte prolactin was grosslyreduced.

Overall, the performance of lambs grazing on AR514 pasture was similarto that on the endophyte-free pasture. No tremors or “ryegrass staggers”symptoms were observed.

TABLE 3 Performance of lambs grazing endophyte infected and endophytefree pasture. Endophyte treatment AR514 (in Year Kentucky 31) Jesup EF*Jesup EI* Live weight gain 1997 103 a** 102 a 67 b (g/hd/d) 1998  93 a102 a 57 b Body temperature 1997  39.8 a  39.8 a 40.1 b (° C.) Bloodprolactin 1997 414 a 400 a Not detectable (b) (ng/ml) 1998 550 a 150 a<0.5 b *EF = endophyte free; EI = infected with common toxic or wildtype endophyte. **Treatments with no letter in common are significantlydifferent (P < 0.05).Endophytes and Resistance to Lesion Nematodes

In some environments, notably sandy soils with relatively warm and humidclimates, nematode may cause significant damage to tall fescue rootsystems thus affecting the persistence of the grass in the pasture. Agreenhouse trial with three plants and nine replicates per treatment hasdemonstrated that endophyte infection may confer resistance to lesionnematodes, Pratylenchus spp. In a greenhouse experiment nematodereproduction was investigated with two tall fescue cultivars infectedwith various endophytes or endophyte free.

Table 4 indicates that an endophyte of this invention, AR514, conferspartial resistance to lesion nematodes to a greater degree thanendophyte-free plants (EF) or two endophytes lacking production ofergovaline (AR542 & AR584) although not to the same degree as the commonendophyte (EI) of the cultivars.

TABLE 4 Endophyte effect on lesion nematode numbers Fescue Endophytecultivar EF* AR542 AR584 AR514 EI* GA 5 146 149 101 — 19 Jesup 147  88120 69 30 Mean 146 a** 118 ab 111 ab 69 b 24 c Relative to 100 a  81 ab 76 ab 47 b 16 c EF = 100 *EF = endophyte free; EI = infected withcommon toxic or wild type endophyte. **Treatments with no letter incommon are significantly different (P < 0.05).Endophytes Confer Resistance to Root Aphid

Endophyte infection is known to affect infestation of grasses by aphids.Table 5 compares treatments of fourteen plants each of tall fescuecultivar Kentucky 31 for mean numbers of root aphids wherein AR514infection is shown to confer considerable protection in comparison withanother endophyte-infected set of plants or endophyte free plants.

TABLE 5 Log number of root aphid per 10 ml sub-sample in Kentucky 31tall fescue. Endophyte treatment No. root aphid No. root aphid/gm rootEndophyte Free 4.043 a* 2.055 a AR542 1.710 b 0.473 b AR514 0.765 c0.095 c *Treatments with no letter in common are significantly different(P < 0.01)Endophytes Confer Deterrence to Corn Flea Beetle

Tall fescue Kentucky 31 leaves infected with examples of the endophytesof this invention (E+) and also leaves infected with wild type toxicendophyte were compared with endophyte free (EF) material using the cornflea beetle Chaetocnema pulicaria in a feeding preference testexperiment. Endophytes AR512, AR513, AR514, AR524 and AR525 allconferred resistance or feeding deterrence similar to that of leavesinfected with the wild type toxic endophyte. The mean of feeding scoresfor the examples of this invention were E+=0.4 as against EF=2.8(P<0.001) on a scale of 0 to 3 (where 0 is no feeding and 3 is extensivefeeding). Score counts of feeding scars (E+=2.2, EF=27.8, mean of thetotal number of scars across 3 transects per leaf blade) and of faecalpellets (E+=9.2, EF=75.8, mean number of faecal pellets on each blade)were also highly significantly different with those for E+ closelycomparable to those of toxic wild type endophyte of Kentucky 31.

A similar single preference test with endophyte AR512 infected intomeadow fescue cultivar Ensign demonstrated an even more extremepreference effect with feeding scores of E+=0.0, EF=3.0, feeding scarsscore counts E+=0, EF=33, and faecal pellets E+=3, EF=50.

Aspects of the present invention have been described by way of exampleonly and it should be appreciated that modifications and additions maybe made thereto without departing from the scope thereof.

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1. A biologically pure culture of an endophyte of the Neotyphodiumcoenophialum species, selected from the group consisting of: AR512;AR513; AR514; AR517; AR521; AR522; AR524; AR525; AR535; AR539; andcombinations thereof; AR512; AR513; AR514; AR517; AR521; AR522; AR524;AR525; AR535, AR539 being cultures deposited on 2 Oct. 2002 at theAustralian Government Analytical Laboratories (AGAL) with accessionnumbers: NM02/31935; NM02/31936; NM02/31937; NM02/31938; NM02/31939;NM02/31940; NM02/31941; NM02/31 942; NM02/31 943; NM02/3 1944,respectively; characterised in that when the endophyte is combined witha host grass, the endophyte does not produce alkaloid compounds atlevels associated with toxicosis in animals; and further characterisedin that when the endophyte is combined with a host grass, the endophyteproduces at least two clavine alkaloids selected from the groupconsisting of: agroclavine; setoclavine; isosetoclavine; andcombinations thereof.
 2. The endophyte culture as claimed in claim 1characterised in that the endophyte does not produce alkaloid compoundsat levels associated with fescue toxicosis.
 3. The endophyte culture asclaimed in claim 1 characterised in that the endophyte does not produceergovaline alkaloid at a level associated with toxicosis.
 4. Theendophyte culture as claimed in claim 3 characterised in that theendophyte produces a level of ergovaline that is less than 0.4 ppm indry matter in herbage consumed by grazing animals.
 5. The endophyteculture as claimed in claim 3, characterised in that the endophyteproduces a level of ergovaline that is less than 0.4 ppm in dry matterin herbage, other than the crown of the host grass, consumed by grazinganimals.
 6. The endophyte culture as claimed in claim 1 characterised inthat the endophyte produces sufficient levels of at least two clavinealkaloids to protect the endophyte and the host grass from pests orabiotic stresses or both.
 7. The endophyte culture as claimed in claim 6characterised in that the clavine alkaloids protect the endophyte andhost grass from abiotic stresses wherein the abiotic stress is a waterdeficit.
 8. A biologically pure endophyte culture of Neotyphodiumcoenophialum selected from the group consisting of an endophyte culturedeposited with the Australian Government Analytical Laboratories underaccession number NM02/31935; NM02/31936; NM02/31937; NM02/31938;NM02/31939; NM02/31940; NM02/31941; NM02/31942; NM02/31943; andNM02/31944.